Cytotoxic antibody directed against type b lymphoid hematopoietic proliferations

ABSTRACT

The present invention relates to a monoclonal antibody directed against the CD20 antigen, wherein the variable region of each of the light chains thereof is encoded by a sequence which shares at least 70% identity with murine nucleic acid sequence SEQ ID No. 5, the variable region of each of the heavy chains thereof is encoded by a sequence which shares at least 70% identity with murine nucleic acid sequence SEQ ID No. 7, and the constant regions of light and heavy chains thereof are constant regions from a non-murine species, as well as for activation of FcγRIIIA receptors in immune effector cells, and for the manufacture of a drug especially for the treatment of leukaemia or lymphoma.

This application is a Continuation of copending application Ser. No.11/793,138, filed on Aug. 14, 2008, which was filed as PCT InternationalApplication No. PCT/FR2005/003123 on Dec. 14, 2005, which claims thebenefit under 35 U.S.C. §119(a) to Patent Application No. 0413320, filedin FRANCE on Dec. 15, 2004, all of which are hereby expresslyincorporated by reference into the present application.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

This application includes an electronically submitted sequence listingin .txt format. The .txt file contains a sequence listing entitled“20151014_(—)3493_(—)0456PUS2_ST25.txt” created on Oct. 14, 2015 and is56,116 bytes in size. The sequence listing contained in this .txt fileis part of the specification and is hereby incorporated by referenceherein in its entirety.

The present invention relates to a monoclonal antibody directed againstthe CD20 antigen, in which the variable regions of each of the lightchains are encoded by sequences which share at least 70% sequenceidentity with murine nucleic acid sequence SEQ ID No. 5, the variableregion of each of the heavy chains are encoded by sequences which shareat least 70% identity with murine nucleic acid sequence SEQ ID No. 7,and the constant regions of the light and heavy chains are constantregions from a non-murine species, as well as to the use of such anantibody for activating FcγRIII receptors in immune effector cells andfor the manufacture of a drug, in particular for the treatment ofleukaemia or lymphoma.

INTRODUCTION AND PRIOR ART

The CD20 antigen is a hydrophobic transmembrane protein with a molecularweight of 35-37 kDa which is present on the surface of mature Blymphocytes (Valentine et al. (1987) Proc. Natl. Acad. Sci. USA 84(22):8085-8089; Valentine et al. (1989) J. Biol. Chem., 264(19):11282-11287). It is expressed during the development of B lymphocytecells (B cells) as from the early pre-B stage until differentiation intoplasmocytes, a stage at which this expression disappears. The CD20antigen is present on both normal B lymphocytes and malign B cells. Morespecifically, the CD20 antigen is expressed on most phenotype-Blymphomas (80% lymphomas): for example, it is expressed on over 90%non-Hodgkin's B-cell lymphomas (NHL) and over 95% B-type ChronicLymphocytic Leukaemia (B-CLL). The CD20 antigen is not expressed onhaematopoietic stem cells and on plasmocytes.

The function of CD20 has not yet been fully clarified, but it may act asa calcium channel and be involved in the regulation of the first stagesof B lymphocytes differentiation (Golay et al. (1985) J. Immunol.135(6): 3795-3801) and proliferation (Tedder et al. (August 1986) Eur.J. Immunol. 16(8): 881-887).

Therefore, although some uncertainty remains as regards its role in theactivation and proliferation of B cells, the CD20 antigen is, because ofits location, an important target for the treatment of conditions whichinvolve tumoural B cells, such as NHL or B-CLL for instance, usingantibodies which specifically recognise CD20. Furthermore, this antigenis an ideal target since it is a membrane protein for which noexpression modulation or polymorphism is known.

Only one non-radioactively labelled anti-CD20 monoclonal antibody,Rituxan® (rituximab, Genentech), is currently commercially available forthe treatment of B-cell lymphoma. It shows encouraging clinical resultsin patients with NHL when associated with chemotherapy. Itseffectiveness, however, remains variable and frequently modest when itis used alone (reeling et al. (2004) Blood 104(6): 1793-1800).

In addition, Rituxan® has only a modest effect on B cells in B-CLL. Thislow degree of effectiveness has been correlated with various phenomena:on the one hand, B-CLL B cells only express CD20 in relatively lowquantities, and on the other hand, Rituxan® only induces very low ADCC(Antibody-Dependent Cellular Cytotoxicity) activity levels against thesecells in vitro. These two observations might explain the correlationthat has been observed between the level of expression of CD20 ontumours (in quantitative flow cytometry) and response to treatments.

Since B-CLL is the commonest form of leukaemia in western countries, andhigh-dose chemotherapy treatment sometimes prove to be insufficient andinvolve side effects which lead to haematopoietic aplasia andimmunodeficiency, monoclonal antibodies appear to be an innovativeapproach. It is therefore of primary importance to develop antibodieswhich are capable of specifically targeting the CD20 antigen and whichallow tumour cells such as B-CLL, which only express this antigen to alimited degree, to be destroyed.

Antibodies 2F2 and 7D8, proposed by Genmab (Teeling et al. (2004) Blood104(6): 1793-1800) for the treatment of B-CLL, have a capacity toactivate the complement which is greater than that induced by Rituxan®.These antibodies, however, have a low ADCC activity, similar to that ofRituxan®. Yet, some clinicians have shown that the complement activityis the cause of deleterious side effects, as the antibodies trigger anactivation system which leads to the production of molecules (inparticular, anaphylatoxins) which have a wide spectrum of non-specificactivities (inflammatory, allergic or vascular reactions etc.). Inaddition, these antibodies are still at the research stage and theirclinical effectiveness has yet to be evaluated.

In application FR03/02713 (WO 2004/029092), the present Applicantdescribes an anti-CD20 antibody which can be produced in the YB2/0 lineand which has been selected for its ability to induce a high ADCCactivity and a high level of IL-2 production by the Jurkat-CD16 cellcompared to Rituxan®. There is a significant need for new anti-CD20antibodies which will allow the range of B-cell diseases treated usingthe currently available immunotherapies to be extended; this isparticularly the case with B-cell diseases in which the CD20 antigen isexpressed to a small degree on the populations of B cells involved, andfor which no satisfactory immunotherapies exist.

It is with this purpose in mind that the present Applicant has developednew CD20 antibodies which exhibit a particularly high ADCC activitycompared to Rituxan®.

DESCRIPTION

A first object of the invention therefore relates to a monoclonalantibody directed against the CD20 antigen, in which the variable regionof each of the light chains is encoded by a sequence which shares atleast 70% identity with murine nucleic acid sequence SEQ ID No. 5, thevariable region of each of the heavy chains is encoded by a sequencewhich shares at least 70% identity with murine nucleic acid sequence SEQID No. 7, and the constant regions of the light and heavy chains areconstant regions from a non-murine species.

The antibodies are made up of heavy and light chains linked together bydisulphide bonds. Each chain is made up, in the N-terminal position, ofa variable region (or domain) (encoded by rearranged V-J genes for thelight chains and V-D-J genes for the heavy chains) specific to theantigen against which the antibody is directed, and, in the C-terminalposition, of a constant region made up from a single CL domain for thelight chains, or several domains for the heavy chains.

For the purposes of the invention, the expressions “monoclonalantibodies” or “monoclonal antibody composition” refer to a preparationof antibody molecules having identical and unique specificities.

The antibody according to the invention, in which the variable regionsin the light and heavy chains are from a species which is different fromthat of the constant regions of the light and heavy chains, is referredto as a “chimeric” antibody.

Murine nucleic acid sequences SEQ ID No. 5 and SEQ ID No. 7 code for thevariable domain of each of the light chains and the variable domain ofeach of the heavy chains respectively, of the antibody produced bymurine hybridoma CAT-13.6E12, available at the Deutsche Sammlung vonMikroorganismen and Zellkulturen GmbH (DSMZ) under number ACC 474. Thishybridoma produces a murine IgG2a,κ-type monoclonal antibody directedagainst CD20.

These murine sequences were chosen to derive the sequences of thevariable regions of the antibodies according to the invention because ofthe specificity of the CAT-13.6E12 murine antibody for the CD20 antigen,the antigen also recognised by Rituxan®. The variable regions of theantibodies according to the invention share at least 70% identity withsequences SEQ ID No. 5 and SEQ ID No. 7, with this sequence identityproviding the antibodies according to the invention with a specificitywhich is identical to that of the CAT-13.6E2 murine antibody.Preferably, this sequence identity also provides the antibody accordingto the invention with an affinity for the target which is identical tothat of the CAT-13.6E12 murine antibody.

In addition, the present Applicant has surprisingly shown that theCAT-13.6E12 murine antibody has the ability to induce the secretion ofIL-2 in the presence of Jurkat-CD16 cells which express ectodomains ofthe FcγRIIIA receptor (as shown in FIG. 11), indicating a strong bindingbetween the murine antibody and human CD16 (FcγRIIIA), which againmotivated the choice made by the present Applicant.

In addition, in the antibodies according to the invention, the constantregions of the light and heavy chains are from a non-murine species. Inthis regard all non-murine mammal species and families may be used, inparticular humans, monkeys, murine (apart from mice), porcine, bovine,equine, feline, canine, as well as birds.

The antibodies according to the invention may be constructed usingstandard recombinant DNA techniques, well known to those skilled in theart, and more particularly using the “chimeric” antibody constructiontechniques described, for example, in Morrison et al. (1984) Proc. Natl.Acad. Sci. USA 81: 6851-6855, where the recombinant DNA technology isused to replace the constant region of a heavy chain and/or the constantregion of a light chain in an antibody from a non-human mammal, with thecorresponding regions in an human immunoglobulin. One particularembodiment will be illustrated below.

Advantageously, the variable region of each of the light chains of theantibody according to the invention is encoded by a sequence whichshares at least 80% identity with murine nucleic acid sequence SEQ IDNo. 5, and the variable region of each of the heavy chains of theantibody according to the invention is encoded by a sequence whichshares at least 80% identity with murine nucleic acid sequence SEQ IDNo. 7.

Advantageously, the variable region of each of the light chains of theantibody according to the invention is encoded by a sequence whichshares at least 90% identity with murine nucleic acid sequence SEQ IDNo. 5, and the variable region of each of the heavy chains of theantibody according to the invention is encoded by a sequence whichshares at least 90% identity with murine nucleic acid sequence SEQ IDNo. 7.

Advantageously, the variable region of each of the light chains of theantibody according to the invention is encoded by a sequence whichshares at least 95% identity with murine nucleic acid sequence SEQ IDNo. 5, and the variable region of each of the heavy chains of theantibody according to the invention is encoded by a sequence whichshares at least 95% identity with murine nucleic acid sequence SEQ IDNo. 7.

Advantageously, the variable region of each of the light chains of theantibody according to the invention is encoded by a sequence whichshares at least 99% identity with murine nucleic acid sequence SEQ IDNo. 5, and the variable region of each of the heavy chains of theantibody according to the invention is encoded by a sequence whichshares at least 99% identity with murine nucleic acid sequence SEQ IDNo. 7.

Advantageously, the invention also relates to any antibody in which thevariable regions of the heavy and light chains include one or moresubstitution(s), insertion(s) or deletion(s) of one or more amino acids,with these sequence modifications complying with the sequence identitypercentage levels defined above, without affecting the antibodys'specificity or affinity for the target.

The antibodies of the invention are also any antibody which includes theCDRs (Complementary Determining Regions) of the CAT-13.6E12 antibody,combined with FR (framework) regions (highly conserved regions of thevariable regions, also known as “backbone” regions). Such antibodieshave affinities and specificities which are closely comparable with, andpreferably identical to, those of the CAT-13.6E12 murine antibody.

Preferably, the variable region of each of the light chains of theantibody according to the invention is encoded by murine nucleic acidsequence SEQ ID No. 5 or by murine nucleic acid sequence SEQ ID No. 25,and the variable region of each of the heavy chains of the antibodyaccording to the invention is encoded by murine nucleic acid sequenceSEQ ID No. 7.

In one embodiment of the invention, an antibody according to theinvention is therefore a monoclonal antibody directed against the CD20antigen, in which the variable region of each of the light chains isencoded by murine nucleic acid sequence SEQ ID No. 5, the variableregion of each of the heavy chains is encoded by murine nucleic acidsequence SEQ ID No. 7, and the constant regions of the light and heavychains are constant regions from a non-murine species.

In a second embodiment, the antibody according to the invention istherefore a monoclonal antibody raised against the CD20 antigen, inwhich the variable regions of each of the light chains are encoded bymurine nucleic acid sequence SEQ ID No. 25, the variable regions of eachof the heavy chains are encoded by murine nucleic acid sequence SEQ IDNo. 7, and the constant regions of the light and heavy chains areconstant regions from a non-murine species.

In both embodiments, the antibodies differ in their nucleotide sequencesby a single nucleotide: the nucleotide located at position 318 in SEQ IDNo. 5 and SEQ ID No. 25, which correspond to a cytosine and an adeninerespectively.

The antibodies of the invention according to these embodiments havespecificities and affinities for the target antigen, CD20, which arecomparable with, and preferably identical to, those of the CAT-13.6E12murine antibody.

Preferably, the constant regions of each of the light chains and each ofthe heavy chains of the antibody according to the invention are humanconstant regions. In this preferred embodiment of the invention, theimmunogenicity of the antibody is reduced in humans, and consequently,the antibodys' effectiveness is improved upon therapeutic administrationto humans.

According to a preferred embodiment of the invention, the constantregion of each of the light chains of the antibody according to theinvention is of x type. Any allotype is suitable for the implementationof the invention, e.g. Km(1), Km(1, 2), Km(1, 2, 3) or Km(3), but thepreferred allotype is Km(3).

According to another additional embodiment, the constant region of eachof the light chains of the antibody according to the invention is of Atype.

According to one specific aspect of the invention, and in particularwhen the constant regions of each of the light chains and of each of theheavy chains of the antibody according to the invention are humanregions, the constant region of each of the heavy chains of the antibodyis of γ type. According to this alternative, the constant region of eachof the heavy chains of the antibody may be of γ1, γ2 or γ3 type, withthese three constant region types exhibiting the specific feature ofbinding the human complement, or even of γ4 type. Antibodies which haveγ-type constant regions for each of the heavy chains belong to the IgGclass. Immunoglobulins G (IgG) are heterodimers made up of 2 heavychains and 2 light chains, linked together by disulphide bonds. Eachchain is made up, in the N-terminal position, of a variable region ordomain (encoded by rearranged V-J genes for the light chains and V-D-Jgenes for the heavy chains) specific to the antigen against which theantibody is directed, and, in the C-terminal position, of a constantregion made up of a single CL domain for the light chain, or of 3domains (CH₁, CH₂ and CH₃) for the heavy chain. Combining the variabledomains and the CH₁ and CL domains of the heavy and light chains make upthe Fab fragments which are linked to the Fc regions through a highlyflexible hinge region allowing each Fab fragment to bind its antigentarget whilst the Fc region, the mediator for the effector properties ofthe antibody, remains accessible to effector molecules such as FcγR andC1q receptors. The Fc region, made up of both CH₂ and CH₃ globulardomains, is glycosylated at the CH₂ domain, with a lactosamine-typebiantennary N-glycan linked to Asn 297 being present on each of the 2chains.

Preferably, the constant region of each of the heavy chains of theantibody is of γ1 type, as such antibody exhibits the ability to induceADCC activity in the greatest number of (human) individuals. In thisrespect, any allotype is suitable for the implementation of theinvention, e.g. G1m(3), G1m(1, 2, 17), G1m(1, 17) or G1m(1, 3).Preferably, the allotype is G1m(1, 17).

According to one particular aspect of the invention, the constant regionof each of the heavy chains of the antibody is of γ1 type, and isencoded by human nucleic acid sequence SEQ ID No. 23, with the constantregion of each of the light chains being encoded by human nucleic acidsequence SEQ ID No. 21. Such an antibody therefore includes a murinevariable region and a human constant region, with γ1-type heavy chains.This antibody therefore belongs to the IgG1 sub-class. According to theembodiment of the antibody according to the invention, the antibody hastwo light chains, the variable domain of which is encoded by murinenucleic acid sequence SEQ ID No. 5 or murine nucleic acid sequence SEQID No. 25, and the human constant region of which is encoded by nucleicacid sequence SEQ ID No. 21, and two heavy chains, the variable domainof which is encoded by murine nucleic acid sequence SEQ ID No. 7 and theconstant region of which is encoded by human nucleic acid sequence SEQID No. 23.

Preferentially, each of the light chains of the antibody according tothe invention is encoded by murine-human chimeric nucleic acid sequenceSEQ ID No. 13 or by murine-human chimeric nucleic acid sequence SEQ IDNo. 27, and each of the heavy chains is encoded by murine-human chimericnucleic acid sequence SEQ ID No. 19. Murine-human chimeric nucleic acidsequence SEQ ID No. 13, which codes for each of the light chains of theantibody, is obtained by fusing murine nucleic acid sequence SEQ ID No.5, which codes for the variable domain of each of the light chains ofthe antibody, to human nucleic acid sequence SEQ ID No. 21, which codesfor the constant region of each of the light chains of the antibody.

Murine-human chimeric nucleic acid sequence SEQ ID No. 27, which codesfor each of the light chains of the antibody, is obtained by fusingmurine nucleic acid sequence SEQ ID No. 25, which codes for the variabledomain of each of the light chains of the antibody, to human nucleicacid sequence SEQ ID No. 21, which codes for the constant region of thelight chains of the antibody.

Murine-human chimeric nucleic acid sequence SEQ ID No. 19, which codesfor each of the heavy chains of the antibody, is obtained by fusingmurine nucleic acid sequence SEQ ID No. 7, which codes for the variabledomain of each of the heavy chains of the antibody, to human nucleicacid sequence SEQ ID No. 23, which codes for the constant region of eachof the heavy chains of the antibody.

According to a particular aspect of the invention, when each of thelight chains of the antibody is encoded by murine-human chimeric nucleicacid sequence SEQ ID No. 13, and each of the heavy chains is encoded bymurine-human chimeric nucleic acid sequence SEQ ID No. 19, the peptidesequence of each of the light chains, deduced from nucleic acid sequenceSEQ ID No. 13, is sequence SEQ ID No. 14 and the peptide sequence ofeach of the heavy chains, deduced from nucleic acid sequence SEQ ID No.19, is sequence SEQ ID No. 20.

According to a further particular aspect of the invention, when each ofthe light chains of the antibody is encoded by murine-human chimericnucleic acid sequence SEQ ID No. 27, and each of the heavy chains isencoded by murine-human chimeric nucleic acid sequence SEQ ID No. 19,the peptide sequence of each of the light chains, deduced from nucleicacid sequence SEQ ID No. 27, is sequence SEQ ID No. 28, and the peptidesequence of each of the heavy chains, deduced from nucleic acid sequenceSEQ ID No. 19, is sequence SEQ ID No. 20.

The peptide sequences SEQ ID No. 14 and SEQ ID No. 28 differ only by theamino acid present at position 106 on each of these two sequences. Theamino acid located at position 106 is lysine (K) in sequence SEQ ID No.28; it is asparagine (N) in sequence SEQ ID No. 14.

The invention also relates to antibodies in which each of the lightchains encoded by murine-human chimeric nucleic acid sequence shares atleast 70% homology or identity with murine-human chimeric nucleic acidsequence SEQ ID No. 13, and each of the heavy chains encoded by amurine-human chimeric nucleic acid sequence shares at least 70% homologyor identity with the murine-human chimeric nucleic acid sequence SEQ IDNo. 19, with these modifications adversely impairing neither thespecificity of the antibody nor its effector activities, such as ADCC(Antibody-Dependent Cell-mediated Cytotoxicity) activity.

In a particularly advantageous manner, the antibody of the invention isproduced by a rat hybridoma cell line. The line which produces theantibody according to the invention is an important characteristic sinceit provides the antibody with certain of its particular properties. Infact, the method of expression of the antibody induces thepost-translational modifications, in particular the glycosylationmodifications, which may vary from one cell line to another, andtherefore provides antibodies which have identical primary structureswith different functional properties.

In a preferred embodiment, the antibody is produced in the rat hybridomaYB2/0 cell line (cell YB2/3HL.P2.G11.16Ag.20, registered at the AmericanType Culture Collection under ATCC number CRL-1662). This line waschosen because of its ability to produce antibodies with improved ADCCactivity compared to antibodies with the same primary structuresproduced, for example, in CHO cells.

According to a specific embodiment, a preferred antibody according tothe invention is antibody EMAB6 produced by clone R509, registered on 8Nov. 2004 under registration number CNCM I-3314 at the CollectionNationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 ruedu Docteur Roux, 75724 Paris Cedex 15, France). Each of the light chainsof the EMAB6 antibody is encoded by murine-human chimeric nucleic acidsequence SEQ ID No. 13, and each of the heavy chains is encoded bymurine-human chimeric nucleic acid SEQ ID No. 19. This chimeric antibodycompetes with the CAT-13.6E12 murine antibody in binding CD20 and has acytotoxic activity which is much greater than that of Rituxan®, whichmay be attributable in part to the specific glycosylation of theN-glycan of the heavy chain of these antibodies (see Example 4). Infact, a specific feature of the R509 clone is that it produces an EMAB6antibody composition with a fucose/galactose ratio of less than 0.6,which has been shown, in patent application FR 03 12229, to be optimalto provide the antibody with strong ADCC activity. This antibody istherefore particularly interesting as a therapeutic tool for thetreatment of conditions in which the cells to be targeted express CD20.

In a further specific embodiment, another preferred antibody accordingto the invention is antibody EMAB603 produced by clone R603, registeredon 29 Nov. 2005 under registration number CNCM 1-3529 at the CollectionNationale de Cultures de Microorganismes (CNCM, Institut Pasteur, 25 ruedu Docteur Roux, 75724 Paris Cedex 15, France). Each of the light chainsof the EMAB603 antibody is encoded by murine-human chimeric nucleic acidsequence SEQ ID No. 27, and each of the heavy chains is encoded bymurine-human chimeric amino acid sequence SEQ ID No. 19. This chimericantibody competes with the CAT-13.6E12 murine antibody in binding CD20and has a cytotoxic activity which is much greater than that ofRituxan®, which may be attributable in part to the specificglycosylation of the N-glycan of the heavy chain of these antibodies(see Example 3). In fact, a specific feature of the R603 clone is thatit produces an EMAB603 antibody composition with a fucose/galactoseratio of less than 0.6, (see Example 4) which has been shown, in patentapplication FR 03 12229, to be optimal to provide the antibody withstrong ADCC activity. This antibody is therefore of particular interestas a therapeutic tool for the treatment of conditions in which the cellsto be targeted express CD20.

Another object of the invention relates to vector pEF-EMAB6-K for theexpression of the light chain of an antibody according to the invention,having sequence SEQ ID No. 12. This vector is the vector which allows anantibody according to the invention, the light chain of which is encodedby nucleic acid sequence SEQ ID No. 13, the deduced peptide sequence ofwhich is sequence SEQ ID No. 14, to be expressed. This vector is anucleic acid molecule into which murine nucleic acid sequence SEQ ID No.5, which codes for the variable domain of each of the light chains ofthe antibody, and nucleic acid sequence SEQ ID No. 21, which codes forthe constant regions of each of the light chains of the antibody, havebeen inserted to be introduced and maintained in a host cell. It allowsforeign nucleic acid fragments to be expressed in the host cell since ithas sequences (promoter, polyadenylation sequence, selection gene) whichare essential to this expression. Such vectors are well known to thoseskilled in the art and may be, without implied limitation, anadenovirus, a retrovirus, a plasmid or a bacteriophage. In addition, anymammalian cell may be used as a host cell, that is as a cell whichexpresses the antibody according to the invention, e.g. YB2/0, CHO, CHOdhfr- (e.g. CHO DX B11, CHO DG44), CHO Lec13, SP2/0, NSO, 293, BHK orCOS.

Another object of the invention relates to vector pEF-EMAB6-H for theexpression of the heavy chain of an antibody according to the invention,having sequence SEQ ID No. 18. This vector is the vector which allows anantibody according to the invention, the heavy chain of which is encodedby nucleic acid sequence SEQ ID NO 19, the deduced peptide sequence ofwhich is sequence SEQ ID No. 20, to be expressed. This vector is anucleic acid molecule into which murine nucleic acid sequence SEQ ID No.7, which codes for the variable domain of each of the heavy chains ofthe antibody, and human nucleic acid sequence SEQ ID No. 23, which codesfor the constant region of each of the heavy chains of the antibody,have been inserted to be introduced and maintained in a host cell. Itallows these foreign nucleic acid fragments to be expressed in the hostcell since it has sequences (promoter, polyadenylation sequence,selection gene) which are essential to this expression. Just asindicated earlier, the vector may be, for example, a plasmid, anadenovirus, a retrovirus or a bacteriophage, and the host cell may beany mammalian cell, e.g. YB2/0, CHO, CHO dhfr- (CHO DX B11, CHO DG44),CHO Lec13, SP2/0, NSO, 293, BHK or COS.

An antibody produced by co-expressing the pEF-EMAB6-K and pEF-EMAB6-Hvectors in the YB2/0 cell is illustrated by the anti-CD20 EMAB6antibody, produced by clone R509 (registered under registration numberCNCM 1-3314 at the CNCM). This antibody induces a cytotoxicity which isgreater than that induced by Rituxan®, both in cells from patients withB-CLL (on which the CD20 antigen is expressed at lower levels) and inRaji cells used as a model and which express CD20 at higher densitiescompared to the cells from patients with B-CLL. Furthermore, the EMAB6antibody induces a secretion of IL-2 (interleukin 2) in Jurkat-CD16cells which is at much higher levels than with Rituxan®. Since the EMAB6antibody can be produced by growing the R509 clone in a culture mediumand under conditions which allow the vectors described above to beexpressed, it is therefore a very interesting tool for advancing thetherapy and diagnosis of B-cell diseases in which the CD20 antigen isinvolved, more specifically B-CLL, as well as for research in this area.

A particular object of the invention is a stable cell line whichexpresses an antibody according to the invention.

Advantageously, the stable cell line which expresses an antibodyaccording to the invention is selected from the group consisting of:SP2/0, YB2/0, IR983F, a human myeloma such as Namalwa or any other cellof human origin such as PERC6, the CHO lines, in particular CHO-K-1,CHO-Lec10, CHO-Lec1, CHO-Lec13, CHO Pro-5, CHO dhfr- (CHO DX B11, CHODG44), or other lines chosen from Wil-2, Jurkat, Vero, Molt-4, COS-7,293-HEK, BHK, K⁶H⁶, NSO, SP2/0-Ag 14 and P3X63Ag8.653.

The line used is preferably the rat hybridoma YB2/0 cell line. This linewas chosen because of its ability to produce antibodies with improvedADCC activity with respect to antibodies with the same primary structureproduced, for example, in CHO cells.

According to one particular aspect of the invention, the stable cellline which expresses an antibody according to the invention and which ismore specifically chosen from the group described above, hasincorporated the two pEF-EMAB6-K and pEF-EMAB6-H expression vectors asdescribed earlier.

One specific aspect of the invention relates to clone R509, registeredunder registration number CNCM 1-3314 at the Collection Nationale deCultures de Microorganismes (CNCM).

One specific aspect of the invention relates to clone R603, registeredunder registration number CNCM 1-3529 at the Collection Nationale deCultures de Microorganismes (CNCM).

Another object of the invention relates to a DNA fragment havingsequence SEQ ID No. 19 which codes for the heavy chain of an antibodyaccording to the invention. Murine-human chimeric nucleic acid sequenceSEQ ID No. 19 codes for each of the heavy chains of the antibody. It isobtained by fusing murine nucleic acid sequence SEQ ID No. 7, whichcodes for the variable domain of each of the heavy chains of theantibody, to human nucleic acid sequence SEQ ID No. 23, which codes forthe constant region of each of the heavy chains of the antibody.

Another specific object of the invention relates to a DNA fragmenthaving sequence SEQ ID No. 13, which codes for the light chain of anantibody according to the invention. Murine-human chimeric nucleic acidsequence SEQ ID No. 13 codes for each of the light chains of theantibody. It is obtained by fusing murine nucleic acid sequence SEQ IDNo. 5, which codes for the variable domain of each of the light chainsof the antibody, to human nucleic acid sequence SEQ ID No. 21, whichcodes for the constant region of each of the light chains of theantibody.

Another specific object of the invention relates to a DNA fragmenthaving sequence SEQ ID No. 27, which codes for the light chain of anantibody according to the invention. Murine-human chimeric nucleic acidsequence SEQ ID No. 27, codes for each of the light chains of theantibody. It is obtained by fusing murine nucleic acid sequence SEQ IDNo. 25, which codes for the variable domain of each of the light chainsof the antibody, to human nucleic acid sequence SEQ ID No. 21, whichcodes for the constant region of each of the light chains of theantibody.

One specific object of the invention relates to the use of the antibodyaccording to the invention to activate, in vivo or in vitro, theFcγRIIIA receptors of effector immune cells. In fact, the antibodies ofthe invention have the specific feature and advantage of beingcytotoxic. As such, they exhibit the ability to activate FcγRIIIAreceptor with their Fc regions. This is of considerable interest as thisreceptor is expressed on the surface of cells known as “effector cells”:binding of the Fc region of the antibody to its receptor carried by theeffector cell causes the activation of FcγRIIIA receptors and thedestruction of the target cells. Effector cells are, for instance, NK(Natural Killer) cells, macrophages, neutrophils, CD8 lymphocytes, Tγδlymphocytes, NKT cells, eosinophils, basophils or mastocytes.

In one specific aspect, the antibody of the invention is used as a drug.Advantageously, such a drug is intended for the treatment of conditionsin which the target cells are cells which express CD20, such asmalignant B-cell lymphoma.

According to one advantageous aspect, the antibody according to theinvention is used to manufacture a drug for the treatment of leukaemiaor lymphoma.

One specific object of the invention is the use of the antibodyaccording to the invention for the manufacture of a drug for thetreatment of a pathology selected from the group consisting of acute Blymphoblastic leukaemia, B-cell lymphoma, mature B-cell lymphoma,including B-type Chronic Lymphocytic Leukaemia (B-CLL), small B-celllymphoma, B-cell prolymphocytic leukaemia, lymphoplasmocytic lymphoma,mantle cell lymphoma, follicular lymphoma, marginal zone MALT-typelymphoma, lymph node marginal zone lymphoma with or without monocytoid Bcells, splenic marginal zone lymphoma (with or without villouslymphocytes), tricholeucocytic leukaemia, diffuse large B-cell lymphoma,Burkitt's lymphoma, as well as any immune dysfunction diseases involvingB lymphoid cells, including auto-immune diseases.

Another object of the invention is the use of the antibody according tothe invention for the manufacture a drug for the treatment of lymphoidleukaemia.

Another object of the invention is the use of the antibody according tothe invention for the manufacture of a drug for the treatment of B-typeChronic Lymphoid Leukaemia (B-CLL). Furthermore, the antibody accordingto the invention is particularly well suited for the treatment ofconditions in which CD20 is less strongly expressed on B cells, andpreferably, B-CLL (B-type Chronic Lymphocytic Leukaemia). In thisregard, the antibody according to the invention may be used incombination with one or more further antibody(ies), e.g. monoclonalantibodies directed against one or more further antigens expressed onlymphoid cells, such as, for example, antigens HLA-DR, CD19, CD23, CD22,CD80, CD32 and CD52, for the manufacture of a drug for the treatment ofleukaemia or lymphoma. Thus, the humanised antibody Campath-1H®(alentuzumab, MabCampathR®) which targets a molecule which is abundantlyexpressed on lymphoid cells, the CD52 antigen, and which induces celllysis by mobilising the host effector mechanisms (complement binding,antibody-dependent cytotoxicity) is used in the treatment of CLL(Moreton P., Hillmen P. (2003) Semin. Oncol. 30(4): 493-501; Rawstron A.C. et al, (2004) Blood 103(6): 2027-2031; Robak T. (2004) Leuk. Lymphoma45(2): 205-219; Stanglmaier M. et al, (2004) Ann. Hematol. 83(10):634-645). Clinical tests are also underway to evaluate antibodies orimmunotoxins which target the antigens HLA-DR, CD22, CD23, CD80 inpatients with CLL (Mavromatis B. H., Cheson B. D. (2004) Blood Rev.18(2): 137-148; Mavromatis B., Cheson B. D. (2003) J. Clin. Oncol.21(9): 1874-1881, Coleman M. et al, (2003) Clin. Cancer Res. 9:3991S-3994S; Salvatore G. et al, (2002) Clin. Cancer Res. 8: 995-1002).

In a further embodiment, the antibody according to the invention may beused in combination with cells which express FcγRs, such as NK cells,NKT (Natural Killers T) cells, Tγδ lymphocytes, macrophages, monocytesor dendritic cells, i.e. in combination with a cellular therapy (PellerS., Kaufman S. (1991) Blood 78: 1569, Platsoucas C. D. et al, (1982) J.Immunol. 129: 2305; Kimby E. et al, (1989) Leukaemia 3(7): 501-504;Soorskaar D. et al, Int. Arch. Allery Appl. Immunol. 87(2): 159-164;Ziegler H. W. et al, (1981) Int. J. Cancer 27(3): 321-327; Chaperot L.et al, (2000) Leukaemia 14(9): 1667-1677; Vuillier F., Dighiero G.(2003) Bull. Cancer. 90(8-9): 744-750).

In addition, the antibody according to the invention advantageouslyallows the doses administered to patients to be reduced: advantageously,the antibody dose administered to the patient is 2 times, 5 times, oreven 10 times, 25 times, 50 times or particularly advantageously 100times less than a dose of Rituxan®. Advantageously, the antibody doseadministered to the patient is between 2 and 5 times, between 5 and 10times, between 5 and 25 times, between 5 and 50 times, or even between 5and 100 times less than a dose of Rituxan®. Thus, the antibody accordingto the invention, for instance the EMAB6 antibody, may advantageously beadministered at a dose of less than 187.5 mg/m², 75 mg/m², 37.5 mg/m²,15 mg/m², 7.5 mg/m², or particularly advantageously less than 3.75mg/m². The dose administered is advantageously between 187.5 mg/m² and75 mg/m², or between 75 mg/m² and 37.5 mg/m², between 75 mg/m² and 15mg/m², between 75 mg/m² and 7.5 mg/m², or even between 75 mg/m² and 3.75mg/m².

Thus, the invention also refers to a method for treating diseases inwhich the target cells are cells which express CD20, such as malignantB-cell lymphoma, consisting in administering to a patient an effectivedose of a composition containing an antibody according to the invention.More specifically, the treatment method is particularly suited to thetreatment of leukaemia or lymphoma. Even more specifically, it is amethod for treating a pathology chosen from acute B lymphoblasticleukaemia, B-cell lymphoma, mature B-cell lymphoma, including B-typeChronic Lymphocytic Leukaemia (B-CLL), small B-cell lymphoma, B-cellprolymphocytic leukaemia, lymphoplasmocytic lymphoma, mantle celllymphoma, follicular lymphoma, marginal zone MALT-type lymphoma, lymphnode marginal zone lymphoma with or without monocytoid B cells, splenicmarginal zone lymphoma (with or without villous lymphocytes),tricholeucocytic leukaemia, diffuse large B-cell lymphoma, Burkitt'slymphoma, as well as any immune dysfunction diseases involving cells ofthe B lymphoid lines, including auto-immune diseases, consisting inadministering an effective dosage of an antibody or antibody compositionaccording to the invention.

A particular object of the invention is the use of an antibody accordingto the invention for the manufacture of a drug for the treatment ofchronic graft-versus-host disease in order to treat symptoms whichinvolve the receiver's B cells.

Finally, a last object of the invention is the use of an antibodyaccording to the invention for the manufacture of a drug for a treatmentof organ, in particular kidney, transplant rejection.

Recent studies (Ratanatharathorn et al, (August 2003) Biol. Blood MarrowTransplant 9(8): 505-511; Becker et al, (June 2004) Am. J. Transplant.4(6): 996-1001) have in fact shown the benefits of anti-CD20 antibodiesin the treatment of such conditions.

Further aspects and advantages of the invention will be described in thefollowing examples which should be regarded as illustrative examples anddo not limit the scope of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the CKHu vector used for thechimerisation of the light chain kappa of antibodies EMAB6 and EMAB603.

FIG. 2: Schematic representation of the light chain pEF-EMAB6-Kexpression vector used for the production of antibody EMAB6.

FIG. 3: Schematic representation of the G1Hu vector used for thechimerisation of the heavy chain of antibodies EMAB6 and EMAB603.

FIG. 4: Schematic representation of the heavy chain pEF-EMAB6-Hexpression vector used for the production of antibody EMAB6.

FIG. 5: Competition by the chimeric EMAB6 antibody for the binding ofthe murine antibody produced by CAT-13.6E12 (CAT13) to CD20 expressed onRaji cells.

FIG. 6A: Complement-dependent cytotoxic activity of the anti-CD20antibodies on Raji cells. Rituxan®: open triangle, EMAB6: closedlozenge. Cell lysis is estimated by measuring the intracellular LDHreleased into the supernatant. Results are expressed as percentagelysis, with 100% being the value obtained with Rituxan® (at 5,000 ng/mLanti-CD20 antibody). Mean of 5 tests. FIG. 6B: Complement-dependentcytotoxic activity of the anti-CD20 antibodies on Raji cells. Comparisonof the complement-dependent cytotoxic activities of EMAB6 (closedlozenge) and EMAB603 (open lozenge).

FIG. 7A: ADCC activity induced by anti-CD20 antibodies in the presenceof Raji cells. Rituxan®: open triangle, EMAB6: closed lozenge. Celllysis is estimated by measuring the intracellular LDH released into thesupernatant. Results are expressed as percentage lysis, with 100% beingthe value obtained with Rituxan® (at 250 ng/mL anti-CD20 antibody). Meanof 3 tests. FIG. 7B: ADCC activity induced by anti-CD20 antibodies inthe presence of Raji Cells. Comparison of ADCC induced by EMAB6 (closedlozenge) and EMAB603 (open lozenge).

FIG. 8: ADCC activity induced by anti-CD20 antibodies in the presence ofB lymphocytes from patients with B-CLL. Rituxan®: open triangle, EMAB6:closed lozenge. E/T ratio=15. Cell lysis is estimated by measuring thecalcein released into the supernatant. Results are expressed aspercentages, with 100% being the value obtained with Rituxan® (at 500ng/mL anti-CD20 antibody). Mean of 4 experiments corresponding to 4different patients.

FIG. 9A: Activation of CD16 (FcγRIIIA) induced by anti-CD20 antibodiesin the presence of Raji cells. Rituxan®: open triangle, EMAB6: closedlozenge. Results are expressed as percentage of IL-2, as measured insupernatants using ELISA; with 100% being the value obtained withRituxan® (at 2,500 ng/mL anti-CD20 antibody). Mean of 4 tests. FIG. 9B:Activation of CD16 (Fcg RIIIA) induced by anti-CD20 antibodies in thepresence of Raji cells. Comparison between the activation of CD16(FcγRIIIA) as induced by EMAB6 (closed lozenge) and EMAB603 (openlozenge).

FIG. 10: Activation of CD16 (FcγRIIIA) induced by anti-CD20 antibodiesin the presence of B lymphocytes from patients with B-CLL. Rituxan®:open triangle, EMAB6: closed lozenge. Results are expressed aspercentage of IL-2, as measured in the supernatants using ELISA; with100% being the value obtained with Rituxan® (at 2,500 ng/mL anti-CD20antibody). Mean of 12 patients.

FIG. 11: Production of IL-2 induced by the CAT-13.6E12 murine antibodyin the presence of Jurkat-CD16 cells (FcγRIIIA).

FIG. 12: Schematic representation of the heavy chain and light chainpRSV-HL-EMAB603 expression vector used for the production of antibodyEMAB603.

EXAMPLES Example 1 Construction of expression vectors for anti-CD20Chimeric Antibodies EMAB6 and EMAB603 A. Determination of the Sequenceof the Variable Regions of the CAT-13.6E12 Murine Antibody

Total RNA from murine hybridoma CAT-13.6E12 cells (supplier: DSMZ, ref.ACC 474), which produces an IgG2a,κ-type immunoglobulin, was isolated(RNAeasy kit, Qiagen ref. 74104). After reverse transcription, thevariable domains of the light (Vκ) and heavy (VH) chains of theCAT-13.6E12 antibody were amplified using the 5′RACE technique (RapidAmplification of cDNA Ends) (GeneRacer kit, Invitrogen ref. L1500-01).The primers used for the two steps were the following:

1. Reverse Transcription Primers

a. Murine Kappa Specific Antisense Primer (SEQ ID No. 1)

5′-ACT GCC ATC AAT CTT CCA CTT GAC-3′b. Murine G2a Specific Antisense Primer (SEQ ID No. 2)

5′-CTG AGG GTG TAG AGG TCA GAC TG-3′

2. 5′RACE PCR Primers

a. Murine Kappa Specific Antisense Primer (SEQ ID No. 3)

5′-TTGTTCAAGAAGCACACGACTGAGGCAC-3′b. Murine G2a Specific Antisense Primer (SEQ ID No. 4)

5′-GAGTTCCAGGTCAAGGTCACTGGCTCAG-3′

The resulting VH and Vκ PCR products were cloned into vectorpCR4Blunt-TOPO (Zero blunt TOPO PCR cloning kit, Invitrogen, ref.K2875-20) and sequenced. The nucleotide sequence of the Vκ region of themurine CAT-13.6E12 antibody is shown as sequence SEQ ID No. 5 and thededuced peptide sequence is sequence SEQ ID No. 6. The Vκ gene belongsto the Vκ4 class [Kabat et al. (1991) “Sequences of Proteins ofImmunological Interest”. NIH Publication 91-3242].

The nucleotide sequence of the VH region of CAT-13.6E12 is sequence SEQID No. 7 and the deduced peptide sequence is sequence SEQ ID No. 8. TheVH gene belongs to the VH1 class [Kabat et al. (1991) “Sequences ofProteins of Immunological Interest”. NIH Publication 91-3242].

B. Construction of Heavy and Light Chain Expression Vectors for ChimericAntibodies EMAB6 and EMAB603 1. Light Chain Kappa Vector 1.1. LightChain Vector for Antibody EMAB6

The Vκ sequence cloned into the pCR4Blunt-TOPO sequencing vector wasamplified using the following cloning primers:

a) Vκ Sense Primer (SEQ ID No. 9)

5′-CTCAGTACTAGT GCCGCCACC ATGGATTTTCAAGTGCAGATTTTC AG-3′

The underlined sequence corresponds to the SpeI restriction site, thesequence in bold lettering corresponds to a Kozak consensus sequence,the ATG initiator is in italics.

b) Vκ Antisense Primer (SEQ ID No. 10)

5′-TGAAGA CACTTGGTG CAGCCACAGTCCGGTTTATTTCCAGCCTGG T-3′

This primer joins the murine Vκ sequences (in italics) to the humanconstant region (Cκ) (in bold). The underlined sequence corresponds tothe DraIII restriction site.

The resulting Vκ PCR product contains the sequence which codes for thenatural signal peptide of the CAT-13.6E12 murine antibody. This Vκ PCRproduct was then cloned between the SpeI and DraIII sites of the lightchain chimerisation vector (FIG. 1), which corresponds to sequence SEQID No. 11, at 5′ in the human constant region Cκ, the nucleic acidsequence of which is sequence SEQ ID No. 21 and the deduced peptidesequence of which is sequence SEQ ID No. 22. The human Cκ sequence ofthis chimerisation vector had been modified beforehand by silentmutagenesis in order to create a DraIII restriction site to allowcloning of murine Vκ sequences to take place. This chimerisation vectorcontains an RSV promoter and a bGH (bovine Growth Hormone)polyadenylation sequence together with the dhfr (dihydrofolatereductase) selection gene.

The light chain sequence of the chimeric EMAB6 antibody encoded by thisvector is shown as SEQ ID No. 13 for the nucleotide sequence andcorresponds to the deduced peptide sequence SEQ ID No. 14.

1.2. Light Chain Vector for Antibody EMAB603

The protocol is the same as for the light chain vector for the EMAB6antibody (see Example 1, B-1.1), apart from the Vκ antisense primerwhich is:

b′) Vκ Antisense Primer (SEQ ID No. 29)

5′-TGAAGA CACTTGGTG CAGCCACAGT CCG

TTTATTTCCA GCCTGGT-3′

This primer joins the murine Vκ sequences (in italics) to the humanconstant region (Cκ) (in bold). The underlined sequence corresponds tothe DraIII restriction site.

This primer also introduces the mutation AAC->AAA (framed nucleotide inthe antisense primer sequence SEQ ID No. 29), which corresponds tomutation N106K (see nucleotide sequence and deduced peptide sequence SEQID No. 25 and SEQ ID No. 26) relative to the natural Vκ sequence ofCAT-13.6E12 (see. SEQ ID No. 5 and SEQ ID No. 6).

The light chain sequence of the chimeric EMAB603 antibody encoded bythis vector is shown as SEQ ID No. 27 for the nucleotide sequence andcorresponds to the deduced peptide sequence SEQ ID No. 28.

2. Heavy Chain Vector

A similar approach was applied to the chimerisation of the heavy chainsof the EMAB6 and EMAB603 antibodies.

The VH sequence cloned into the pCR4Blunt-TOPO vector was first of allamplified using the following cloning primers:

a) VH Sense Primer (SEQ ID No. 15)

5′-CTCAGTACTAGT GCCGCCACC ATGGGATTCAGCAGGATCTTTCT C-3′

The underlined sequence corresponds to the restriction site SpeI, thesequence in bold lettering corresponds to a Kozak consensus sequence,the ATG initiator is in italics.

b) VH Antisense Primer (SEQ ID No. 16)

5′-GACCGAT GGGCCC TTGGTGGAGGCTGAGGAGACGGTGACTGAGGTT CC-3′

This primer joins the murine VH sequences (in italics) to the human G1constant region (in bold). The underlined sequence corresponds to theApaI restriction site.

The amplified VH fragment contains the sequence which codes for thenatural signal peptide of the CAT-13.6E12 murine antibody. This VH PCRproduct was then cloned between the SpeI and ApaI sites in the heavychain chimerisation vector (FIG. 3) which corresponds to sequence SEQ IDNo. 17, at 5′ of the γ1 human constant region, the nucleic acid sequenceof which is sequence SEQ ID No. 23 and the deduced peptide sequence ofwhich is sequence SEQ ID No. 24. This chimerisation vector contains anRSV promoter and a bGH (bovine Growth Hormone) polyadenylation sequenceas well as the neo selection gene.

The heavy chain sequences of the chimeric EMAB6 and EMAB603 antibodiesencoded by this vector are shown as SEQ ID No. 19 for the nucleotidesequence and as SEQ ID No. 20 for the deduced peptide sequence.

3. Final Expression Vectors 3.1. EMAB6 Antibody Expression Vectors

For the expression of the EMAB6 antibody, the RSV promoter of the kappalight chain chimerisation vector (see Example 1, B-1.1) was replacedwith the human EF-1 alpha promoter. The final light chain pEF-EMAB6-Kexpression vector is shown in FIG. 2 and corresponds to sequence SEQ IDNo. 12.

The light chain sequence of the chimeric EMAB6 antibody encoded by thisvector is shown as SEQ ID No. 13 for the nucleotide sequence andcorresponds to the deduced peptide sequence SEQ ID No. 14.

For the expression of the EMAB6 antibody, the RSV promoter of the heavychain chimerisation vector (see Example 1, B-2) was replaced with thehuman EF-1 alpha promoter. The thus-obtained final heavy chainpEF-EMAB6-H expression vector is shown in FIG. 4 and corresponds tosequence SEQ ID No. 18.

3.2. EMAB603 Antibody Expression Vector

A unique expression vector containing both heavy chain and light chaintranscription units of the anti-CD20 EMAB603 antibody was constructedfrom two light and heavy chain chimerisation vectors (see Example 1,B-1.2 and B2 respectively) by sub-cloning into the XhoI site of theheavy chain vector, a BglII-PvuII fragment of the light chain vectorcontaining the light chain transcription unit and the dhfr gene. ThispRSV-HL-EMAB603 expression vector includes two selection genes, i.e. neo(neo-phosphotransferase II) and dhfr (dihydrofolate reductase), togetherwith two heavy chain and light chain transcription units under thecontrol of an RSV promoter (FIG. 12).

Example 2 Production of Cell Lines Derived from the YB2/0 Line ProducingAnti-CD20 Chimeric EMAB6 and EMAB603 Antibodies

The rat YB2/0 cell line (ATCC # CRL-1662) was cultivated in EMS medium(Invitrogen, ref. 041-95181M) containing 5% foetal calf serum (JRHBiosciences, ref. 12107). For transfection, 5 million cells wereelectroporated (Biorad electroporator, model 1652077) in Optimix medium(Equibio, ref. EKITE 1) with 25 μg of light chain vector pEF-EMAB6-K(FIG. 2), linearised with AatII, and 27 μg of heavy chain vectorpEF-EMAB6-H (FIG. 4), linearised with ScaI, for the expression of theEMAB6 antibody, or with vector pRSV-HL-EMAB603, for the expression ofthe EMAB603 antibody. The electroporation conditions applied were 230Volts and 960 microFarads in a 0.5-mL cuvette. Each electroporationcuvette was then distributed over 5 P96 plates at a density of 5,000cells/well.

Placement in a selective RPMI medium (Invitrogen, ref 21875-034)containing 5% dialysed serum (Invitrogen, ref. 10603-017), 500 μg/mLG418 (Invitrogen, ref. 10131-027) and 25 nM methotrexate (Sigma, ref.M8407), was carried out 3 days after transfection.

The supernatants from the resistant transfection wells were screened forthe presence of chimeric immunoglobulin (Ig) by applying an ELISA assayspecific to the human Ig sequences.

The 10 transfectants producing the largest amount of antibody wereamplified on P24 plates and their supernatants re-assayed using ELISA toestimate their productivity and select, by limited dilution (40cells/plate), the best three producers for cloning.

After cloning, the R509.6A4 clone (R509-33903/046-6H1(1)6A4,productivity: 17 μg/10⁶ cells), hereafter referred to as “R509”, as wellas the R603 clone were selected for the production of the chimeric EMAB6and EMAB603 antibodies respectively and progressively acclimated to theCD Hybridoma production medium (Invitrogen, ref. 11279-023).

The production of the chimeric EMAB6 and EMAB603 antibodies was achievedby expanding, in CD Hybridoma medium, the acclimated culture obtained bydilution to 3×10⁵ cells/mL in 75-cm² and 175-cm² vials and then dilutionto 4.5×10⁵ cells/mL in roller flasks. Once the maximum volume (1 L) wasachieved, culture was continued until the cell viability was only 20%.After production, the chimeric EMAB6 and EMAB603 antibodies werepurified using protein-A affinity chromatography (HPLC estimatedpurity<95%) and checked by polyacrylamide gel electrophoresis.

Example 3 Characterisation of the Functional Activity of ChimericAntibodies EMAB6 and EMAB603 A. Specificity

Specificity of the antigen recognition of the chimeric EMAB6 antibodywas evaluated by studying the competition with the murine antibodyCAT-13.6E12 (CAT13) for binding the CD20 antigen expressed by Rajicells.

For that purpose, the EMAB6 antibody (10 μL at 0.5 to 50 μg/mL) wasincubated at 4° C. with a fixed quantity of CAT-13.6E12 murine antibody(10 μL at 5 μg/mL) for 20 minutes in the presence of Raji cells (50 μLat 4×10⁶ cells/mL). After washing, a mouse anti-IgG antibody coupled tophycoerythrin (PE) was added to the Raji cells so as to specificallydetect the binding of the CAT-13.6E12 murine antibody. The MedianFluorescence Intensities (MFIs) obtained in the presence of variousconcentrations of EMAB6 are converted to percentages, with 100%corresponding to binding to CAT-13.6E12 cells in the absence of theEMAB6 antibody.

An inhibition curve is thus obtained for binding of the CAT-13.6E12(CAT13) antibody to Raji cells in the presence of increasingconcentrations of EMAB6 (FIG. 5).

This study demonstrates that the chimerisation process has not adverselyaffected the specificity of the EMAB6 antibody, which does compete withthe parental CAT-13.6E12 murine antibody for binding to CD20 expressedon the surface of Raji cells.

The antigen recognition specificity of the EMAB603 antibody iscomparable with that of the EMAB6 antibody.

B. Complement-Dependent Cytotoxic Activity

Complement-dependent cytotoxic activity of the EMAB6 and EMAB603antibodies was examined with Raji cells in the presence of young rabbitserum as a source of complement; the anti-CD20 chimeric antibodyRituxan® was included in one test, for comparison.

For this test, the Raji cells were adjusted to 6×10⁵ cells/mL in IMDM(Iscove's Modified Dulbecco's Medium) 5% FCS (Foetal Calf Serum). Theantibodies were diluted with IMDM+0.5% FCS. The reaction mixture wasmade up of 50 μL antibody, 50 μL young rabbit serum (1/10 IMDM+0.5% FCSdilution of Cedarlane CL 3441 reagent), 50 μL target cells and 50 μLIMDM+0.5% FCS medium. The final antibody concentrations were 5,000,1,250, 250 and 50 ng/mL. A control without antibodies was included inthe test. After 1 hr incubation at 37° C. in a 5% CO₂ atmosphere, theplates were centrifuged and the levels of intracellular LDH releasedinto the supernatant estimated using a specific reagent (CytotoxicityDetection Kit 1 644 793).

The percentage lysis was estimated using a calibration range obtainedusing various dilutions of target cells lysed using triton X100 (2%)corresponding to 100, 50, 25, and 0% lysis respectively.

The results shown in FIG. 6(A) demonstrate that EMAB6 and Rituxan® bothinduce complement-dependent lysis of the Raji cells. Nevertheless, EMAB6complement activity appeared to be slightly less than that of Rituxan®.This difference is greater at the low concentrations of antibody used inthis test. Thus for concentrations of 50 and 250 ng/mL, the activity ofEMAB6 is of the order of 45% of that of Rituxan®. This differencebecomes smaller as the antibody concentration is increased, with the %complement-dependent cytotoxic activity of the EMAB6 antibodyrepresenting 92% of that of Rituxan® at the highest concentrationtested, i.e. 5,000 ng/mL.

This lower complement-dependent cytotoxic activity of the AMAB6 antibodycompared to that of Rituxan® may be regarded as an advantage, since itlimits the potential in vivo toxicity of EMAB6 compared to Rituxan®,associated with the activation of the conventional complement pathway,which leads to the production of various molecules with undesirableinflammatory, allergic and vascular activities.

The complement activity of the EMAB603 antibody is shown in FIG. 6(B).

C. ADCC Activity

The cytotoxicity of the chimeric EMAB6 antibody was evaluated in thepresence of Raji cells or B lymphocytes from patients with CLL. Theanti-CD20 chimeric antibody Rituxan® was included in the tests forcomparison.

The calcein-labelling ADCC measurement technique used was as follows:

NK cells were isolated from PBMCs using the separation on magnetic beads(MACS) technique from Myltenyi. The cells were washed and re-suspendedin IMDM+5% FCS (45×10⁵ cells/mL). The effector cells and target cellswere used in a ratio of 15/1. The Raji cells or the PBMCs (PeripheralBlood Mononuclear Cells) from patients with B-CLL obtained after Ficolltreatment (>95% B cells) were labelled beforehand with calcein (1 mLcells at 3×10⁶ cells/mL in IMDM+5% FCS+20 μL calcein (20 μM), 20 minincubation at 37° C. and then washing with HBSS (Hank's Buffered SalineSolution)) and adjusted to 3×10⁵ cells/mL in IMDM+5% FCS. The antibodieswere diluted with IMDM+0.5% FCS (final concentrations: 500; 50; 5; 0.5;0.05 and 0.005 ng/mL).

The reaction mixture was made up of 50 μL antibody, 50 μL effectorcells, 50 μL target cells and 50 μL IMDM medium in a P96 microtitrationplate. Two negative controls were used:

-   -   Lysis without NK: NK effector cells were replaced with IMDM+5%        FCS.    -   Lysis without antibodies (Ab): antibodies were replaced with        IMDM+5% FCS.

After 4 hrs incubation at 37° C. in a 5% CO₂ atmosphere, the plates werecentrifuged and the fluorescence associated with the supernatant wasmeasured using a fluorimeter (excitation: 485 nm, emission: 535 nm).

The percentage lysis was estimated using a calibration range obtainedusing various dilutions of target cells lysed using Triton X100 (2%),corresponding to 100, 50, 25, and 0% lysis respectively.

The results were first calculated using the following formula:

% lysis=(% lysis with Antibody and NK)−(% lysis without Antibody)−(%lysis without NK)

and then expressed as relative percentages, with 100% being the valueobtained at the highest concentration of Rituxan®.

The results obtained for the EMAB6 antibody on the Raji line cells shownin FIG. 7(A) demonstrate that, irrespective of the concentration beingtested, the cytotoxicity induced by the EMAB6 antibody is greater thanthat induced by Rituxan®. This difference is particularly large at lowantibody concentrations. Thus, at 0.5 ng/mL, the lysis percentages were96% and 4% for EMAB6 and Rituxan® respectively. By increasing the dose500-fold (250 ng/mL), the difference is still appreciable since therelative percentages of ADCC are 164% and 100% for EMAB6 and Rituxan®respectively. When the EC50s were calculated (antibody concentrationcorresponding to 50% of the E Max, the maximum effectiveness obtained atthe highest antibody concentration and at the plateau) by graphicalestimation (in ng/mL) and assuming that Rituxan® and EMAB6 attain thesame E Max, the Rituxan® EC50/EMAB6 EC50 ratio in this test was thenequal to 300.

The cytotoxicity of the chimeric EMAB603 antibody was evaluated in thepresence of Raji cells using the same procedure as for the EMAB6antibodies. Its activity was comparable with that of the EMAB6 antibody(see FIG. 7(B)).

With the lymphocytes from patients with B-CLL, the results obtained,shown in FIG. 8, demonstrate that, irrespective of the concentrationbeing tested, the cytotoxicity induced by the EMAB6 antibody is greaterthan that induced by Rituxan®. As already observed with the Raji cells,this difference is particularly large at low antibody concentrations. Aconcentration of 0.5 ng/mL EMAB6 induces the same percentage lysis as500 ng/mL Rituxan®, i.e. a concentration ratio of 1,000. At 5 ng/mL, thelysis percentages are 269% and 9% for EMAB6 and Rituxan® respectively.At the maximum dose tested (500 ng/mL), the difference is still verylarge since the relative percentages of ADCC are 350% and 100% for EMAB6and Rituxan® respectively. An interesting result corresponds to theconcentrations which give rise to 50% lysis. In this test, the Rituxan®EC50/EMAB6 EC50 ratio was estimated as 10,000 (graphical estimate inng/mL for EC50 assuming that Rituxan® and EMAB6 attain the same E Max).

In these tests, the cytotoxic activities of EMAB6 and EMAB603 aretherefore much greater than that of Rituxan®.

D. Activation of CD16 (IL-2 Secretion)

The activation of CD16 (FcγRIIIA) induced by the chimeric EMAB6 antibodywas determined in the presence of Raji cells or B lymphocytes frompatients with CLL. This test evaluated the ability of the antibody tobind to CD16 (FcγRIIIA) receptor expressed on the Jurkat-CD16 cells andto induce the secretion of IL-2. The anti-CD20 chimeric antibodyRituxan® is included in the tests for comparison.

Measurement of CD16 activation was carried out in the following manneron the Jurkat-CD16 cell line in the presence of Raji cells or Blymphocytes from patients with CLL.

Mixture in 96-well plate: 50 μL antibody solution (dilution to 10,000,1,000, 100 and 10 ng/mL with IMDM+5% FCS for B lymphocytes from patientswith B-CLL and 10,000, 2,000, 1,000, 200, 100, 50 and 25 ng/mL for Rajicells), 50 μL PMA (Phorbol Myristate Acetate, diluted to 40 ng/mL withIMDM+5% FCS), 50 μL Raji or PBMCs from patients with B-CLL obtainedafter Ficoll treatment (>95% B cells) diluted to 6×10⁵/mL with IMDM+5%FCS, and 50 μL Jurkat-CD16 cells (20×10⁶/mL in IMDM+5% FCS). Controlswithout antibodies were included in all tests. After incubationovernight at 37° C., the plates were centrifuged and the IL-2 containedin the supernatants estimated using a commercial kit (Quantikine fromR/D). The OD readings were made at 450 nm.

The results were initially expressed as IL-2 levels as a function of theantibody concentration (from 0 to 250 ng/mL final concentration), thenas relative percentages, where 100% is the value obtained with Rituxan®at the highest test concentration.

The results obtained with the Raji line cells shown in FIG. 9(A)demonstrate that, in the presence of EMAB6 and Rituxan®, the Jurkat-CD16cells secrete IL-2, which indicates cell activation via binding of theFc portion of the antibodies to CD16. The EMAB6 antibody, however, hasan inductive activity which is much stronger than the Rituxan® antibody.Thus, at 6.25 ng/mL, the IL-2 percentages were 112% and 21% for EMAB6and Rituxan® respectively. At 50 ng/mL, the difference is still large,with the percentages of IL-2 being 112% and 65% respectively. Thisdifference decreases as concentration increases, with the respectivepercentages of IL-2 for EMAB6 and Rituxan® being 124% and 100% at 2,500ng/mL. In this test, the Rituxan® EC50/EMAB6 EC50 ratio is estimated at15 (graphical estimate in ng/mL for EC50 assuming that Rituxan® andEMAB6 attain the same E Max).

These results confirm the ADCC results, both being CD16-dependant. Theydemonstrate that the binding to CD16 (FcγRIIIA) by the Fc portion of theEMAB6 antibody is followed by a strong cellular activation which leadsto the induction of effector functions.

The activation of CD16 (FcγRIIIA) induced by the chimeric EMAB603antibody in the presence of Raji cells is comparable with that inducedby the EMAB6 antibody.

With lymphocytes from patients with B-CLL, the results obtained shown inFIG. 10 demonstrate that in the presence of anti-CD20 Rituxan® andEMAB6, the Jurkat-CD16 cells secrete IL-2, which indicates cellactivation via binding of the Fc portion of the antibodies to CD16. TheEMAB6 antibody, however, has an inductive ability which is much greaterthan the Rituxan® antibody. In fact, the IL-2 secretion inductionactivity of Rituxan® is close to the base line at concentrations of 2.5and 25 ng/mL, whereas that of the EMAB6 antibody is significant. Thus at25 ng/mL, the IL-2 percentages were 132% and 34% for EMAB6 and Rituxan®respectively. At the highest concentration (2,500 ng/mL), the IL-2percentages were 148% and 100% respectively. The Rituxan® EC50/EMAB6EC50 ratio in this test is greater than 100: it is estimated at 300(graphical estimate in ng/mL for EC50 assuming that Rituxan® and EMAB6attain the same E Max).

In conclusion, all the tests carried out on Raji cells demonstrate thatthe EMAB6 and EMAB603 antibodies, unlike Rituxan®, are highly cytotoxicand induce the activation of cells which express CD16 (FcγRIIIA),especially at low antibody concentrations. On the contrary, under thesame conditions, the complement-dependent cytotoxic activity of EMAB6decreases by about 50% compared to that of Rituxan®.

These results are confirmed by the studies carried out using cellsisolated from patients with B-CLL, suggesting that the EMAB6 antibody ismuch more cytotoxic than Rituxan® towards B lymphocytes from patientswith B-CLL. The differences between the two antibodies are more markedwith lymphocytes from patients with B-CLL than with the Raji cells,which demonstrates the significant therapeutic interest of EMAB6compared to Rituxan® for this condition.

The reason of this increased difference may be, amongst other, the lowerantigen expression of CD20 on B lymphocytes from patients with B-CLLcompared to Raji cells.

By analogy with Raji cells, it may be suggested that thecomplement-dependent cytotoxic activity of the EMAB6 antibody towardslymphocytes from patients with B-CLL must be less than that induced byRituxan®, thus exhibiting the advantage of being less toxic in vivo as aresult of the undesirable effects associated with a strong activation ofthe conventional complement pathway.

Example 4 Analysis of EMAB6 and EMAB603 Glycans by HPCE-LIF

The N-glycan structure of the heavy chains of the EMAB6 and EMAB603antibodies was analysed using HPCE-LIF. The N-glycan structure of theheavy chain of Rituxan® was also analysed for comparison.

For that purpose, anti-CD20 monoclonal antibodies were desalted on aSephadex G-25 column (HiTrap Desalting, Amersham Biosciences),evaporated and re-suspended in the hydrolysis buffer of PNGase F (Glyko)in the presence of 50 mM β-mercaptoethanol. After 16 hrs incubation at37° C., the protein fraction was precipitated by adding absolute ethanoland the supernatant, which contained the N-glycans, was evaporated. Theresulting oligosaccharides were either directly labelled using afluorochrome: APTS (1-amino-pyrene-3,6,8-trisulphonate), or subjected tothe action of specific exoglycosidases before labelling with APTS. Theresulting labelled oligosaccharides were injected onto an N-CHOcapillary, separated and quantified by capillary electrophoresis withlaser-induced fluorescence detection (HPCE-LIF).

The estimation of the fucose level was carried out either by theaddition of the isolated fucosylated forms, or more specifically afterthe simultaneous action of neuraminidase, β-galactosidase andN-acetylhexosaminidase, which resulted in 2 peaks corresponding to thefucosylated or non-fucosylated pentasaccharide [GlcNac2-Man3] beingobtained on the electrophoretogram:

TABLE 1 Analysis of anti-CD20 EMAB603 and Rituxan ® N-glycans Anti-CD20% Fucose % Galactose Fuc/Gal EMAB603 15 37 0.4 Rituxan ® 93 57 1.63

The fucose level, expressed as %, was calculated using the followingformula:

${{Fucose}\mspace{14mu} {level}} = \frac{{{fucosylated}\mspace{14mu}\left\lbrack {{GlcNac}\; 2\text{-}{Man}\; 3} \right\rbrack} \times 100}{\left\lbrack {{{GlcNac}\; 2\text{-}{Man}\; 3} + {{fucosylated}\mspace{14mu} {GlcNac}\; 2\text{-}{Man}\; 3}} \right\rbrack}$

The galactose level, expressed as %, was calculated by adding thepercentages of the oligosaccharide forms containing galactose obtainedafter the action of neuraminidase and fucosidase. The formula used is asfollows:

Galactose level=(G1+G1B)+2x(G2+G2B)

The fucose/galactose ratio is obtained by dividing the fucose level bythe galactose level, calculated as described above.

From this analysis (see Table 1), it appears that the EMAB6 and EMAB603antibodies are little fucosylated (% fucose<25%) compared to Rituxan® (%fucose=93%). In addition, the Fuc/Gal ratio (fucose/galactose ratio) forEMAB6 and EMAB603 is low (Fuc/Gal ratio<0.6), unlike the antibodiesexpressed in CHO cells such as Rituxan® (Fuc/Gal ratio=1.63).

1-39. (canceled)
 40. A method for the treatment of a disease in whichthe target cells are cells which express CD20, which comprisesadministering to a patient an effective amount of a monoclonal antibodydirected against the CD20 antigen, wherein each of the light chainsthereof has the murine-human chimeric amino acid sequence SEQ ID No. 28,and each of the heavy chains thereof has the murine-human chimeric aminoacid sequence SEQ ID No.
 20. 41. The method of claim 40, wherein thedisease in which the target cells are cells which express CD20 is animmune dysfunction disease involving B lymphoid cells.
 42. The method ofclaim 41, wherein the immune dysfunction disease involving B lymphoidcells is selected from auto-immune diseases.
 43. The method of claim 40,wherein the disease in which the target cells are cells which expressCD20 is chronic graft-versus-host disease.
 44. The method of claim 40,wherein the disease in which the target cells are cells which expressCD20 is organ transplant rejection.
 45. The method of claim 44, whereinthe organ transplant rejection is kidney transplant rejection.
 46. Themethod of claim 40, wherein each of the light chains thereof is encodedby murine-human chimeric nucleic acid sequence SEQ ID No. 27, and eachof the heavy chains thereof is encoded by murine-human chimeric nucleicacid sequence SEQ ID No.
 19. 47. The method of claim 40, wherein theantibody is produced by a rat hybridoma cell line.
 48. The method ofclaim 47, wherein the antibody is produced in the rat hybridoma cellline YB2/3HL.P2.G11.16Ag.20, registered at the American Type CultureCollection under ATCC number CRL-1662.
 49. The method of claim 48,wherein the antibody is the EMAB603 antibody produced by clone R603,registered under registration number CNCM 1-3529 at the CollectionNationale de Cultures de Microorganismes (CNCM).
 50. The method of claim40, wherein the antibody is produced in a CHO line.